Modern Pathology (2006) 19, 820–831
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Prognostic significance of dysadherin
expression in epithelioid sarcoma and its
diagnostic utility in distinguishing epithelioid
sarcoma from malignant rhabdoid tumor
Teiyu Izumi1, Yoshinao Oda1, Tadashi Hasegawa2, Yukihiro Nakanishi3, Hiroshi Iwasaki4,
Hiroshi Sonobe5, Hiroaki Goto6, Hidenari Kusakabe7, Tomonari Takahira1,8,
Chikashi Kobayashi1, Ken-ichi Kawaguchi1,8, Tsuyoshi Saito1,8, Hidetaka Yamamoto1,
Sadafumi Tamiya1, Yukihide Iwamoto8 and Masazumi Tsuneyoshi1
1
Department of Anatomic Pathology, Pathological Sciences, Graduate School of Medical Sciences, Kyushu
University, Fukuoka, Japan; 2Department of Clinical Pathology, Sapporo Medical University School of
Medicine, Sapporo, Japan; 3Pathology Division, National Cancer Center Research Institute, Tokyo, Japan;
4
Department of Pathology, School of Medicine, Fukuoka University, Fukuoka, Japan; 5Department of
Pathology, Fukuyama Medical Center, Hiroshima, Japan; 6Department of Pediatrics, Yokohama City
University School of Medicine, Yokohama, Japan; 7Department of Dermatology, Osaka Medical College,
Osaka, Japan and 8Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu
University, Fukuoka, Japan
Dysadherin is a cancer-associated cell membrane glycoprotein, which downregulates E-cadherin and promotes
metastasis. We studied the clinicopathological features in 72 cases of epithelioid sarcoma and in six cases of
malignant rhabdoid tumor, and also assessed the immunohistochemical expression of dysadherin, E-cadherin
and MIB-1 in epithelioid sarcoma and malignant rhabdoid tumor cases. In addition, we compared dysadherin
mRNA expression between epithelioid sarcoma and malignant rhabdoid tumor cell lines, using RT-PCR and realtime quantitative RT-PCR analysis. Immunohistochemical dysadherin expression was more frequently observed
in proximal-type epithelioid sarcoma (71%) in comparison with distal-type epithelioid sarcoma (36%) (P ¼ 0.037).
Furthermore, seven proximal-type epithelioid sarcoma cases mimicking malignant rhabdoid tumor (histologically classified as the large cell type, accompanied by frequent rhabdoid cells and located in deep soft tissue)
were all positive for dysadherin (100%), whereas dysadherin expression was not detected at all in any of the true
six malignant rhabdoid tumors (0%). Cell lines established from proximal-type epithelioid sarcoma revealed
significantly higher levels of dysadherin mRNA expression, compared with the levels seen in malignant rhabdoid
tumor cell lines by real-time quantitative RT-PCR (P ¼ 0.0433). Epithelioid sarcoma patients with dysadherin
expression survived for a significantly shorter time than those without dysadherin expression (P ¼ 0.001). In
multivariate analysis, dysadherin immunopositivity (P ¼ 0.0004) was one of the two independent adverse
prognostic factors. We conclude that dysadherin expression in epithelioid sarcoma is a significant poor
prognostic factor and that it is a powerful diagnostic marker for distinguishing epithelioid sarcoma, including the
proximal-type epithelioid sarcoma, from malignant rhabdoid tumor. In epithelioid sarcoma, especially in
proximal-type epithelioid sarcoma, increased cell disadhesion and motility by dysadherin plays an important role
to acquire aggressive biological behavior. However, in malignant rhabdoid tumor, cell growth cycle that is
regulated by hSNF5/INI1 gene seems to be critical to lethal biological behavior rather than dysadherin.
Modern Pathology (2006) 19, 820–831. doi:10.1038/modpathol.3800599; published online 24 March 2006
Keywords: dysadherin; epithelioid sarcoma; malignant rhabdoid tumor; prognosis; diagnosis; immunohisto-
chemistry
Correspondence: Dr Y Oda, MD, Department of Anatomic
Pathology, Pathological Sciences, Graduate School of Medical
Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka
812-8582, Japan.
E-mail: [email protected]
Received 8 December 2005; revised and accepted 2 March 2006;
published online 24 March 2006
Epithelioid sarcoma was initially described as a
distinct clinicopathological entity by Enzinger in
1970.1 The classical distal-type epithelioid sarcoma
occurs in the distal extremities, mainly in the hand
and around the wrist of young adults. It is known to
be slowly progressive, appearing as multiple, superficial soft-tissue tumors. Epithelioid sarcoma has
Dysadherin in epithelioid sarcoma and MRT
T Izumi et al
821
also been reported to occur in the proximal region of
the extremities and in the trunk, as proximal-type
epithelioid sarcoma.2–4 Malignant rhabdoid tumor
usually occurs in young children across a wide
range of anatomical sites, and this tumor follows a
rapidly and aggressive clinical course. Rhabdoid
cells with intermediate filamentous inclusion
bodies have been found in a large variety of softtissue tumors.5 In particular, one of the most
difficult tumors to distinguish from malignant
rhabdoid tumor is the proximal-type of epithelioid
sarcoma.2,3,6 Some authors7 have described proximal-type epithelioid sarcoma as being a variant of
extra-renal malignant rhabdoid tumor, while other
authors2,8,9 have demonstrated that both tumors are
distinctly different entities. Some data exist regarding the histological and immunohistochemical differences between the proximal-type of epithelioid
sarcoma and malignant rhabdoid tumor, but such
findings have not yet been found to be conclusive.10
In the current study, we compared epithelioid
sarcoma and malignant rhabdoid tumor clinicopathologically and focused on the differences in
dysadherin expression between these two types
of tumor. The aim of this study was to clarify
differences in biological behavior and immunohistochemical profile between the tumors.
lioid sarcoma were classified into three histologic
subtypes. Malignant rhabdoid tumor comprised
three cases of renal type and three cases of extrarenal type. The tumor cells typically had abundant
eosinophilic cytoplasmic inclusions, and the nuclei
were eccentrically located, vesicular, and had
prominent central nucleoli, frequently demonstrating a so-called rhabdoid appearance (Figure 1e and
f). In all six malignant rhabdoid tumor cases, no
other clear alternative line of histological differentiation could be documented. That is the histological difference between proximal-type epithelioid
sarcoma and malignant rhabdoid tumor. Survival
data were available for 71 cases of epithelioid
sarcoma and six cases of malignant rhabdoid tumor.
Follow-up ranged from 1 to 251 months (mean, 50.7
months) in epithelioid sarcoma. All six malignant
rhabdoid tumor patients died of disease at between
1 and 10 months (mean, 6.3 months) after diagnosis.
In order to assess the correlation between clinicopathologic factors and dysadherin expression, the
anatomical site of the tumor, histologic subtype, age,
sex, tumor size, tumor depth, mitotic rate, tumor
necrosis, the presence of rhabdoid cells, hemorrhage
and vascular permeation were analyzed. The
influence of these factors on prognosis has been
previously reported.11
Materials and methods
Immunohistochemistry
Cases and Specimens
Immunohistochemical study was performed using
mouse IgG monoclonal antibodies against dysadherin12 (NCC-M53, 1:1000, National Cancer Center,
Tokyo), E-cadherin (1:1000, BD Transduction
Laboratories, San Jose, CA, USA) and Ki-67 (MIB-1,
1:100, Dako, Grostrup, Denmark). From among the
72 cases of epithelioid sarcoma, 46 cases were
suitable for dysadherin and E-cadherin immunostaining and 52 cases were suitable for MIB-1
immunostaining, whereas in the case of malignant
rhabdoid tumor, all six cases could be studied using
the three kinds of antibodies. Specimens are
pretreated in an autoclave for dysadherin (20 min,
1201C) or in a microwave oven for E-cadherin and
MIB-1 (20 min in both cases). Sections were incubated with the primary antibodies at 41C overnight,
followed by staining with a streptavidin-biotinperoxidase kit (Nichirei, Tokyo, Japan). Staining
for dysadherin was localized to the cell membranes.
The pattern of dysadherin immunostaining in
tumors was compared with that observed in the
basal cells of normal skin tissue, endothelial cells
and lymphocytes. Tumors were classed as dysadherinpositive when more than 50% of the cells were
stained.13–16 The MIB-1-labeling index was estimated as the percentage of Ki-67-positive cells
based on a count of 1000 tumor cells within the
tumor. Dysadherin and E-cadherin expression were
evaluated by four of the authors (TI, YO, TH and
MT) without any knowledge of the clinical features
in each case.
Cases were selected from among more than 12 000
cases of bone and soft-tissue tumors registered in
the Department of Anatomic Pathology, Graduate
School of Medical Sciences, Kyushu University,
Fukuoka, Japan and in the National Cancer Center,
Tokyo, Japan, between 1955 and 2004. As written
informed consent was not obtained, identifying
information for all cases was removed before
analysis for strict privacy protection. There were
72 cases of epithelioid sarcoma, and six cases of
malignant rhabdoid tumor. Subtypes of epithelioid
sarcoma comprised 37 cases of distal-type and 35
cases of proximal-type. We distinguished between
distal-type epithelioid sarcoma and proximal-type
epithelioid sarcoma based on the anatomical site
of the tumor. Histologically, distal-type epithelioid
sarcoma typically shows a nodular or multinodular proliferation of mainly spindle-shaped cells
admixed with epithelioid cells (conventional type)
(Figure 1a and b). In contrast, proximal-type epithelioid sarcoma typically reveals a diffuse proliferation of large epithelioid carcinoma-like cells with
marked cytologic atypia and frequent occurrence of
rhabdoid features (large cell type) (Figure 1c and d).
As described by Hasegawa,8 proximal-type epithelioid sarcoma are classified into three histologic
subtypes (large cell type, conventional type and
angiomatoid type). In the current study, both distaltype epithelioid sarcoma and proximal-type epithe-
Modern Pathology (2006) 19, 820–831
Dysadherin in epithelioid sarcoma and MRT
T Izumi et al
822
Figure 1 Distal-type epithelioid sarcoma typically shows a nodular or multinodular proliferation of mainly spindle-shaped cells
admixed with epithelioid cells (conventional type) (a, b). Proximal-type epithelioid sarcoma typically reveals a diffuse proliferation of
large carcinoma-like epithelioid cells with geographic tumor necrosis (c). Marked cytologic atypia and a rhabdoid cell (black arrow) are
also observed (large cell type) (d). Malignant rhabdoid tumor typically has abundant eosinophilic cytoplasmic inclusions, and the nuclei
are eccentrically located, vesicular, and have prominent central nucleoli, frequently showing a so-called rhabdoid appearance (e, f).
No other clear alternative line of histological differentiation could be documented.
Cell Culture
Four epithelioid sarcoma cell lines (HS-ES-1M,17
YCUS-5,18 ES-OMC-MN,19 SFT860620) and four
malignant rhabdoid tumor cell lines (TM87-16,21
TTC549,22 TC289,23 STM91-0124) were generously
Modern Pathology (2006) 19, 820–831
provided by the co-authors for this study. HS-ES-1M
and YCUS-5 were established from proximaltype epithelioid sarcoma, while ES-OMC-MN and
SFT8606 were established from distal-type epithelioid sarcoma. Human hepatoma Li-7 and PLC/PRF/
5 cell lines were obtained from the Institute of
Dysadherin in epithelioid sarcoma and MRT
T Izumi et al
Development, Aging and Cancer, Tohoku University.
Cells were cultured in RPMI-1640, DMEM or
DMEM/F-12 (GIBCO/BRL) supplemented with 10%
FBS (GIBCO/BRL) in a humidified atmosphere of 5%
CO2 and 95% air.
RT-PCR and Real-Time Quantitative RT-PCR to Detect
Dysadherin mRNA
Total RNA was prepared, using Trizol Reagent
(Invitrogen Corp., Carlsbad, CA, USA) according to
the manufacturer’s protocol. RNA (5 ml) in each
sample were used for the subsequent reverse
transcription. Sequences of specific pairs of primers
were as follows: dysadherin (upper primer: 50 -AGA
GCA CCA AAG CAG CTC AT-30 ; lower primer: 50 GGG TCT GTC TGG ACG TCT GT-30 ; product size,
85 bp). The PCR products were electrophoresed in
2.0% agarose gel and visualized with ethidium
bromide. cDNA from the Li-7 human hepatoma cell
line was used as a positive control for dysadherin
expression, and PLC/PRF/5 was used as a negative
control.12
Real-time quantitative RT-PCR (TaqMan PCR) for
dysadherin was performed using an ABI PRISM
7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and predeveloped
TaqMan assay reagents of human dysadherin and
GAPDH. The PCR reaction was carried out according
to the manufacturer’s protocol.25 The standard curve
was constructed with serial dilutions of the cDNA
samples of Li-7. All reactions of the samples were
triplicated and the data were averaged from the
values obtained in each reaction. To determine the
mRNA levels of dysadherin, we used an mRNA
expression index (EI), which is a relative mRNA
expression level standardized by using the internal
housekeeping gene, GAPDH. The dysadherin mRNA
EI was calculated as follows (in AU): mRNA
EI ¼ (copy numbers of dysadherin mRNA/copy
numbers of GAPDH mRNA) X 1000 AU. A high
(low) level of dysadherin expression was defined as
an mRNA EI above (below) the value of hepatoma
Li-7 cell line.
823
Statistical Analysis
The survival curves were analyzed by the Kaplan–
Meier method and the outcome of different groups
of patients was compared by the log-rank test. The
Cox proportional hazards regression with the stepwise procedure was used in multivariate analysis
of survival data. Fisher’s exact test was used to
evaluate the association between two dichotomous
variables. The correlations between each group and
mRNA expression were determined by using the
Mann–Whitney U test. Probability values of o0.05
were considered as significant.
Results
Clinical and Histological Findings
Epithelioid sarcoma
The clinicopathological findings of 72 cases of
epithelioid sarcoma are summarized in Table 1. In
all, 37 patients were affected at distal sites, whereas
35 patients had a tumor at proximal sites. Histologically, 46 patients were classified as the conventional type, five as the angiomatoid type and 21 as
the large cell type. Rhabdoid cells were detected to
varying degrees in 42 cases (distal-type: 18/37 cases;
proximal-type: 24/35 cases), but were absent in 30
cases. The correlation between the tumor site in all
the epithelioid sarcoma cases and histologic subtype
is summarized in Table 2. The proximal-type often
demonstrated the large cell type (57%: 20/35 cases),
whereas the distal-type predominantly revealed the
non-large cell type (97%: 36/37 cases) and rarely
disclosed the large cell type (3%: 1/37 cases)
(P ¼ 0.0001). The prognosis of the patients with the
proximal-type was significantly worse, when compared with that of patients with the distal-type
(P ¼ 0.019; Figure 2). In contrast, no statistically significant difference in prognosis was seen in histological subtype, among the conventional type, the
angiomatoid type and the large cell type (Figure 3).
The correlation between the clinicopathological
parameters in all the epithelioid sarcoma cases and
Table 1 Clinicopathological parameters in 72 cases of epithelioid sarcoma
Site
Proximal
Distal
Histologic subtype
35
37
Mitotic rate (per 10 HPFs)
Z20
o20
32
40
Conventional
Angiomatoid
Large cell
46
5
21
Tumor necrosis (%)
Z50
o50
0
16
45
11
Age (years)
Z31
o31
32
40
Rhabdoid cells
(+)
()
42
30
Sex
Male
Female
Size (cm)
44
28
Hemorrhage
(+)
()
Unknown
46
20
6
45
r5
Unknown
Depth
31
39
2
Deep
Superficial
22
50
Vascular permeation
(+)
()
57
15
Modern Pathology (2006) 19, 820–831
Dysadherin in epithelioid sarcoma and MRT
T Izumi et al
824
prognosis is summarized in Table 3. In univariate
analysis, proximal tumor site (P ¼ 0.019), male
(P ¼ 0.0285), large tumor size (45 cm) (P ¼ 0.0446),
deep location (P ¼ 0.0457), a high mitotic rate (Z20/
10HPF) (P ¼ 0.0127) and massive tumor necrosis
(Z50%) (P ¼ 0.0049) were all significantly correlated to a worse overall survival rate.
Malignant rhabdoid tumor
Clinicopathological findings of malignant rhabdoid
tumor are summarized in Table 4. There were three
cases of extra-renal malignant rhabdoid tumor and
three cases of renal malignant rhabdoid tumor. The
age of the patients ranged from 0.25 years to 31 years
(mean, 6.8 years). All patients died within 10
months. The prognosis of the patients with malig-
Figure 2 Survival curve of patients with distal-type epithelioid
sarcoma, proximal-type epithelioid sarcoma and malignant
rhabdoid tumor. The prognosis of patients with proximal-type
epithelioid sarcoma was significantly worse than that of patients
with distal-type epithelioid sarcoma (P ¼ 0.019). Moreover, the
prognosis of patients with malignant rhabdoid tumor was
significantly worse than that of patients with proximal-type
epithelioid sarcoma (P ¼ 0.0056).
Figure 3 Survival curve of epithelioid sarcoma patients with the
angiomatoid type, the conventional type and the large cell type.
No statistical significant difference in prognosis was seen among
the three histological subtypes.
Table 3 Clinicopathological parameters and prognosis in epithelioid sarcoma (n ¼ 71)
Table 2 Correlation between tumor site and histologic subtype in
epithelioid sarcoma (n ¼ 72)
Histologic subtype
Site
P-value
Proximal
Non-large cell
Conventional
Angiomatoid
Large cell
15
12
3
20
(43%)
(34%)
(9%)
(57%)
35 (100%)
a
Distal
36
34
2
1
0.0001a
(97%)
(92%)
(5%)
(3%)
37 (100%)
Variables
P-value
Site (proximal)
Histologic subtype (large cell)
Age (Z31 years)
Sex (male)
Size (45 cm, n ¼ 70)
Depth (deep)
Mitotic rate (Z20 per 10 HPFs)
Tumor necrosis (Z50%)
Rhabdoid cells (+)
Hemorrhage (+, n ¼ 65)
Vascular permeation (+)
0.0190a
0.1022
0.0898
0.0285a
0.0446a
0.0457a
0.0127a
0.0049a
0.6279
0.4435
0.2123
a
Statistically significant.
Statistically significant.
Table 4 Clinicopathological parameters in 6 cases of malignant rhabdoid tumor
Site
Extra-renal
Renal
Age (years)
3
3
Mitotic rate (per 10 HPFs)
Z20
420
Modern Pathology (2006) 19, 820–831
5
1
46
r6
Sex
1
5
Tumor necrosis (%)
Z50
o50
0
1
5
0
Male
Female
Size (cm)
3
3
Rhabdoid cells
(+)
()
6
0
45
r5
Depth
3
3
Hemorrhage
(+)
()
6
0
Deep
Superficial
6
0
Vascular permeation
(+)
()
5
1
Dysadherin in epithelioid sarcoma and MRT
T Izumi et al
825
nant rhabdoid tumor was significantly worse, when
compared with that of the patients with proximaltype epithelioid sarcoma (P ¼ 0.0056; Figure 2).
Immunohistochemical Findings in Epithelioid
Sarcoma and Malignant Rhabdoid Tumor
Details of the immunohistochemical findings (dysadherin, E-cadherin and MIB-1) in epithelioid
sarcoma and malignant rhabdoid tumor are summarized in Table 5. We could successfully obtain
dysadherin-positive staining in the basal cells of
normal epidermis, lymphocytes and endothelial
cells of epithelioid sarcoma and malignant rhabdoid
tumor cases, as an internal positive control.12
Dysadherin-positive staining was diffusely and
strongly observed in the membranes of epithelioid
sarcoma cells in 25/46 cases and it was frequently
detected in proximal-type epithelioid sarcoma cases
(Figure 4a), whereas dysadherin expression was
sometimes absent in distal-type epithelioid sarcoma
cases (Figure 4b) but was completely negative in all
six malignant rhabdoid tumor cases (Figure 4c).
There was a statistically significant difference in
dysadherin expression between epithelioid sarcoma
and malignant rhabdoid tumor (P ¼ 0.0232; Table 5).
E-cadherin membranous expression was not observed in any of the 46 cases of epithelioid sarcoma
or in any of the six cases of malignant rhabdoid
tumor. None of the cases showed distinctive
membranous staining, but rather showed cytoplasmic or aberrational nuclear staining (Figure 4d). A
high MIB-1-labeling index (Z30%)26 was observed
in only 29/52 cases (distal-type: 8/23 cases; proximal-type: 21/29 cases) of epithelioid sarcoma, but
in five of six cases of malignant rhabdoid tumor. A
high MIB-1-labeling index (Z30%) was significantly
more frequently observed in proximal-type epithelioid sarcoma (Figure 4e) than in distal-type epithelioid sarcoma (Figure 4f) (P ¼ 0.0072), whereas no
statistically significant difference was seen between
the total cases of epithelioid sarcoma and malignant
Table 5 Dysadherin, E-cadherin and MIB-1 immunoreactivity in
epithelioid sarcoma and malignant rhabdoid tumor
Antibody
Epithelioid
sarcoma
Malignant
rhabdoid tumor
Dysadherin
(+)
()
(n ¼ 46)
25
21
(n ¼ 6)
0
6
E-cadherin
(+)
()
(n ¼ 46)
0
46
(n ¼ 6)
0
6
MIB-1 (%)
Z30
o30
(n ¼ 52)
29
23
(n ¼ 6)
5
1
a
Statistically significant.
rhabdoid tumor (P ¼ 0.3845; Table 5). The correlations between dysadherin immunoreactivity and
clinicopathological parameters in epithelioid sarcoma are summarized in Table 6. Dysadherin expression was significantly more frequently observed in
proximal-type epithelioid sarcoma (17/24 cases),
than in distal-type epithelioid sarcoma (8/22 cases)
(P ¼ 0.037). Male patients (20/29 cases) showed
dysadherin expression significantly more frequently
than female patients (5/17 cases) (P ¼ 0.0144). Other
parameters had no statistically significant correlation with dysadherin expression. Dysadherin immunoreactivity in comparison between malignant
rhabdoid tumor and epithelioid sarcoma mimicking
malignant rhabdoid tumor are summarized in Table
7. Seven proximal-type epithelioid sarcoma cases
and 1 distal-type epithelioid sarcoma case mimicking malignant rhabdoid tumor (histologically classified as the large cell type, accompanied by frequent
rhabdoid cells and located in deep soft tissue) were
all positive for dysadherin (100%), whereas dysadherin expression was not detected at all in any
of the true six malignant rhabdoid tumors (0%).
Dysadherin mRNA Expression in Epithelioid Sarcoma
and Malignant Rhabdoid Tumor
The two epithelioid sarcoma cell lines, HS-ES-1M
and YCUS-5, established from proximal-type epithelioid sarcoma showed higher dysadherin mRNA
expression compared to the two distal-type epithelioid sarcoma cell lines, ES-OMC-MN and SFT8606.
In contrast, all the four malignant rhabdoid tumor
cell lines, TM87-16, TTC549, TC289 and STM91-01
showed no visible dysadherin mRNA expression
(Figure 5). Dysadherin mRNA EIs are summarized in
Table 8. Only the two proximal-type epithelioid
sarcoma cell lines, HS-ES-1M and YCUS-5 showed
very high dysadherin mRNA EIs (4387.9 and 472.20
AU; respectively) compared with the hepatoma Li-7
cell line (40.7 AU). As for the comparison between
epithelioid sarcoma and malignant rhabdoid tumor,
dysadherin mRNA EIs in epithelioid sarcoma
(median, 240.34 AU) were significantly higher than
those in malignant rhabdoid tumor (median, 0.325
AU) (P ¼ 0.0433).
P-value
Prognostic Value of Dysadherin Expression in
Epithelioid Sarcoma
0.0232a
0.3845
Epithelioid sarcoma patients with dysadherin
immunoreactivity survived for a significantly shorter
time than those without dysadherin immunoreactivity (P ¼ 0.001; Figure 6). In addition, distal-type
epithelioid sarcoma patients with dysadherinpositive expression had a significantly worse prognosis than those without dysadherin expression
(P ¼ 0.0059; Figure 7a). Proximal-type epithelioid
sarcoma patients with dysadherin-positive expression had a tendency to have a poor prognosis
Modern Pathology (2006) 19, 820–831
Dysadherin in epithelioid sarcoma and MRT
T Izumi et al
826
Figure 4 Immunohistochemical staining for dysadherin, E-cadherin and MIB-1. Dysadherin membranous staining was frequently
observed in proximal-type epithelioid sarcoma cases (a), whereas dysadherin expression was sometimes absent in distal-type epithelioid
sarcoma (b) and was completely negative in all malignant rhabdoid tumor cases (c). Aberrational nuclear staining for E-cadherin was
detected in proximal-type epithelioid sarcoma cases (d). A high MIB-1-labeling index (Z30%) was frequently seen in proximal-type
epithelioid sarcoma cases (e) (MIB-1: 33%) as compared with distal-type epithelioid sarcoma cases (f) (MIB-1: 4%).
Modern Pathology (2006) 19, 820–831
Dysadherin in epithelioid sarcoma and MRT
T Izumi et al
827
compared with those without dysadherin expression (P ¼ 0.1152; Figure 7b). Multivariate analysis of
the 45 cases for which survival data and immunoTable 6 Correlation between dysadherin immunoreactivity and
clinicopathological parameters in epithelioid sarcoma (n ¼ 46)
Variables
Dysadherin
P-value
histochemical analysis were available revealed that
dysadherin immunopositivity (P ¼ 0.0004) and
massive tumor necrosis (Z50%) (P ¼ 0.0006) were
independent poor prognostic factors (Table 9). Other
clinicopathological prognostic factors were not
significant.
Discussion
(+)
()
Site
Proximal (n ¼ 24)
Distal (n ¼ 22)
17
8
7
14
0.037a
Histologic subtype
Non-large cell (n ¼ 32)
Conventional (n ¼ 27)
Angiomatoid (n ¼ 5)
Large cell (n ¼ 14)
15
11
4
10
17
16
1
4
0.1988
Age (years)
Z31 (n ¼ 27)
o31 (n ¼ 19)
17
8
10
11
0.2314
Sex
Male (n ¼ 29)
Female (n ¼ 17)
20
5
9
12
0.0144a
Size (cm)
45 (n ¼ 23)
r5 (n ¼ 23)
14
11
9
23
0.5544
Depth
Deep (n ¼ 19)
Superficial (n ¼ 27)
12
13
7
14
0.3769
Figure 5 Results of RT-PCR to detect dysadherin mRNA expression in epithelioid sarcoma and malignant rhabdoid tumor cell
lines. P: positive control (Li-7); N: negative control (PLC/PRF/5);
Lanes 1–4: epithelioid sarcoma cell lines; Lanes 5–8: malignant
rhabdoid tumor cell lines. Four epithelioid sarcoma cell lines
revealed high to low levels of dysadherin mRNA expression. The
two proximal-type epithelioid sarcoma cell lines, HS-ES-1 M and
YCUS-5, showed higher dysadherin mRNA expression compared
to the two distal-type epithelioid sarcoma cell lines, ES-OMC-MN
and SFT8606. In contrast, all the four malignant rhabdoid tumor
cell lines showed no visible dysadherin mRNA expression.
Mitotic rate (per 10 HPFs)
Z20 (n ¼ 25)
o20 (n ¼ 21)
16
9
9
12
0.235
Table 8 Dysadherin mRNA expression in epithelioid sarcoma
Tumor necrosis (%)
Z50 (n ¼ 13)
o50 (n ¼ 33)
8
17
5
16
0.7437
Rhabdoid cells
(+) (n ¼ 28)
() (n ¼ 18)
15
10
13
8
40.9999
Hemorrhage
(+) (n ¼ 33)
() (n ¼ 13)
18
7
15
6
40.9999
Vascular permeation
(+) (n ¼ 11)
() (n ¼ 35)
7
18
4
17
0.5138
a
Epithelioid sarcoma and malignant rhabdoid tumor
are both very rare soft-tissue sarcomas and their
and malignant rhabdoid tumor cell lines
Statistically significant.
Dysadherin mRNA EI (AU)
Epithelioid sarcoma cell lines
HS-ES-1 M (proximal-type)
YCUS-5 (proximal-type)
ES-OMC-MN (distal-type)
SFT8606 (distal-type)
4387.90
474.20
8.48
0.52
Malignant rhabdoid tumor cell lines
TM-8716
TTC549
TC289
STM91-01
5.88
0.60
0.05
0.00
Hepatoma cell lines
Li-7
PLC/PRF/5
40.70
1.00
Table 7 Dysadherin immunoreactivity in comparative cases between malignant rhabdoid tumor and epithelioid sarcoma
Histologic subtype
Proximal-type epithelioid sarcoma
Distal-type epithelioid sarcoma
Malignant rhabdoid tumor
Large cell
Large cell
Rhabdoid cells
(+)
(+)
(+)
Depth
Deep
Deep
Deep
Dysadherin
(+)
()
7
1
0
0
0
6
Modern Pathology (2006) 19, 820–831
Dysadherin in epithelioid sarcoma and MRT
T Izumi et al
828
Table 9 Survival analysis in epithelioid sarcoma (n ¼ 45)
Variables
Site (proximal)
Histologic subtype (large cell)
Age (Z31 years)
Sex (male)
Size (45 cm)
Depth (deep)
Mitotic rate (Z20 per 10 HPFs)
Tumor necrosis (Z50%)
Rhabdoid cells (+)
Hemorrhage (+)
Vascular permeation (+)
Dysadherin (+)
MIB-1 (Z30%)
Figure 6 Effect of dysadherin expression on survival in the total
cases of epithelioid sarcoma. Epithelioid sarcoma patients with
dysadherin immunoreactivity survived for a significantly shorter
time than those without dysadherin immunoreactivity (P ¼ 0.001).
Figure 7 Effect of dysadherin expression on survival in distaltype epithelioid sarcoma and proximal-type epithelioid sarcoma.
(a) Distal-type epithelioid sarcoma patients with dysadherinpositive expression had a significantly poor prognosis compared
with those without dysadherin expression (P ¼ 0.0059). (b)
Proximal-type epithelioid sarcoma patients with dysadherinpositive expression had a tendency to have a poor prognosis
compared with those without dysadherin expression, although
the difference was not statistically significant (NS; P ¼ 0.1152).
Modern Pathology (2006) 19, 820–831
a
P-value on survival analysis
Univariate
Multivariate
0.25720
0.40660
0.82870
0.09170
0.0362a
0.09800
0.09080
0.0047a
0.76160
0.89340
0.48120
0.0001a
0.0029a
0.67890
0.48110
0.93550
0.80950
0.53070
0.68640
0.27070
0.0006a
0.45570
0.81070
0.46180
0.0004a
0.27090
Statistically significant.
clinicopathological differences, especially when
comparing proximal-type epithelioid sarcoma with
malignant rhabdoid tumor, are still controversial.
In addition, no valuable prognostic markers for
epithelioid sarcoma and malignant rhabdoid tumor
have been demonstrated in a study of a large
series.
Guillou2 described that proximal-type epithelioid
sarcoma typically demonstrates a diffuse proliferation of large epithelioid cells with marked cytologic
atypia (large cell type), whereas Hasegawa8 classified 20 cases of proximal-type epithelioid sarcoma
into three histologic subtypes (large cell type (60%),
conventional type (30%) and angiomatoid type
(10%)). In the current study, both proximal-type
epithelioid sarcoma and distal-type epithelioid
sarcoma demonstrated three histologic subtypes. In
proximal-type epithelioid sarcoma, 20/35 cases
(57%) were classified as the large cell type, 12/35
cases (34%) as the conventional type, and 3/35 cases
(9%) as the angiomatoid type. Each percentage of
three histologic subtypes was quite similar to that
described by Hasegawa.8 In distal-type epithelioid
sarcoma, 36/37 cases (97%) revealed the non-large
cell type, but the one case (3%) disclosed the large
cell type. Interestingly, Rakheja27 described a case of
epithelioid sarcoma that occurred in the hand of a
14-year-old boy and demonstrated the ‘large cell
type’ morphology. In addition, Guillou2 presented a
case of epithelioid sarcoma in the forearm of 55year-old man with the ‘large cell type’ morphology
(case 18). We considered that the ‘large cell type’
morphology was not restricted to proximal-type
epithelioid sarcoma.
Dysadherin is a cancer-associated cell membrane
glycoprotein, which down-regulates E-cadherin and
promotes metastasis.12 Dysadherin is expressed in a
variety of cancer cells and malignant melanoma
cells.12–16,28 To date, no investigation into dysadherin
expression in sarcomas has been reported.
Dysadherin in epithelioid sarcoma and MRT
T Izumi et al
829
Both epithelioid sarcoma and malignant rhabdoid
tumor are mesenchymal tumors exhibiting epithelial
profiles such as immunohistochemical expression
of cytokeratin29 and epithelial membrane antigen.30
They also possess ultrastructural features such as
specialized junctions, tonofilaments and microvilli
indicative of epithelial differentiation both in vivo31
and in tissue culture.32 In the current study, we are
able to report that immunohistochemical expression
of dysadherin is significantly useful for the differential diagnosis between epithelioid sarcoma and
malignant rhabdoid tumor, and for the prediction of
prognosis in epithelioid sarcoma.
With regard to the differential diagnosis, over 54%
(25/46 cases) of the patients with epithelioid
sarcoma showed dysadherin expression. Proximaltype epithelioid sarcoma (71%; 17/24 cases) showed
significantly frequent dysadherin expression, compared with distal-type epithelioid sarcoma (36%;
8/22 cases). Over the half (15/28 cases) of the
epithelioid sarcoma patients with rhabdoid cells
showed dysadherin expression. In contrast, dysadherin expression was completely negative in
all six malignant rhabdoid tumor cases. Parallel
to the immunohistochemical results, proximal-type
epithelioid sarcoma cell lines showed significantly
higher dysadherin mRNA levels compared to distaltype epithelioid sarcoma or malignant rhabdoid
tumor cell lines. It is sometimes difficult to distinguish epithelioid sarcoma, especially proximaltype epithelioid sarcoma, from malignant rhabdoid
tumor by histological morphology based on hematoxylin–eosin staining. However, it would seem
that immunoreactivity for dysadherin could help
to differentiate between distal-type epithelioid
sarcoma, proximal-type epithelioid sarcoma and
malignant rhabdoid tumor. We have to pay attention
to distinguish epithelioid sarcoma from malignant
rhabdoid tumor, especially when proximal-type
epithelioid sarcoma cases demonstrate the ‘large
cell type’ morphology, accompanied by frequent
rhabdoid cells, and located in deep soft tissue. In the
current study, seven proximal-type epithelioid sarcoma cases and one distal-type epithelioid sarcoma
case mimicking malignant rhabdoid tumor (histologically classified as the large cell type, accompanied
by frequent rhabdoid cells and located in deep soft
tissue) were all positive for dysadherin (100%),
whereas dysadherin expression was not detected at
all in any of the true six malignant rhabdoid tumors
(0%).
In epithelioid sarcoma, Chase and Enzinger
reported that increased patient age, male sex,
proximal tumor location, increased tumor size, high
mitotic rate, tumor necrosis and hemorrhage, and
vascular invasion all had negative prognostic significance.11 Evans and Baer reported that the major
factor relating to distant metastasis and death from
the tumor was the tumor size.33 In the present study,
proximal tumor site, male, large tumor size (45 cm),
deep location, a high mitotic rate (Z20/10HPF) and
massive tumor necrosis (Z50%) were all found to
have a significant relationship to poor prognosis in
univariate analysis.
Dysadherin evokes tumor aggressiveness by increasing cell motility.12,13,16 Dysadherin stimulates
cell motility, and contributes directly to the metastatic potential of human pancreatic cancer cells.13,16
Previous studies have reported that dysadherin
tends to be expressed more frequently in dissociated
tumor cells and infiltrative tumor nests than in welldifferentiated tumor nests.13,16 In the current study,
dysadherin expression was found to be an independent poor prognostic factor by both univariate and
multivariate analysis. In the near future, molecular
targeting therapy for dysadherin may become one of
the first therapeutic choices instead of conventional
therapies in cases of epithelioid sarcoma.
Some previous studies have demonstrated a
significant correlation between dysadherin expression and E-cadherin expression in tongue carcinoma,16 but other studies have failed to demonstrate
this correlation in pancreatic, colorectal, or gastric
carcinoma.13–15 In epithelioid sarcoma and malignant rhabdoid tumor, distinctive membranous expression of E-cadherin was not observed in any
cases, and E-cadherin expression was independent
from dysadherin expression. Smith et al described
the lack of E-cadherin expression in seven cases
of epithelioid sarcoma.34 Saito et al demonstrated
one case of focal membranous and two cases of
membranous and cytoplasmic E-cadherin expression
among 15 cases of epithelioid sarcoma.35 We
reviewed these epithelioid sarcoma cases for the
current study, however, and found that they did not
demonstrate distinctive E-cadherin membranous
expression. We considered and interpreted these
cases to be cases of negative E-cadherin membranous expression. Our result was the same as that
described by Smith et al, accordingly, we consider
that the inactivation of E-cadherin is not caused by
dysadherin alone in epithelioid sarcoma and malignant rhabdoid tumor.
With regard to the prognosis of malignant rhabdoid tumor, no dysadherin expression was observed
and therefore it could not be a prognostic marker.
Malignant rhabdoid tumor is known to have inactivating mutations or deletions of both alleles of
the tumor suppressor gene, hSNF5/INI1 on chromosome 22q11.2.36–38 We consider that the highly
aggressive and lethal biological behavior in malignant rhabdoid tumor is due to abnormalities in this
gene. Interestingly, hSNF5/INI1 gene deletions have
been reported in proximal-type epithelioid sarcoma,
suggesting its involvement in the genesis and/or
progression of proximal-type epithelioid sarcoma.39
Morphological similarities between malignant rhabdoid tumor and proximal-type epithelioid sarcoma
may be partially due to this common genetic
disorder.
In conclusion, dysadherin is frequently expressed
in epithelioid sarcoma, especially in proximal-type
Modern Pathology (2006) 19, 820–831
Dysadherin in epithelioid sarcoma and MRT
T Izumi et al
830
epithelioid sarcoma, whereas it is completely absent
in malignant rhabdoid tumor. Therefore, the immunohistochemical expression of dysadherin is a
significantly useful diagnostic marker for distinguishing proximal-type epithelioid sarcoma from
malignant rhabdoid tumor. Moreover, in epithelioid
sarcoma, dysadherin expression is an independent
adverse prognostic factor, as assessed by multivariate survival analysis.
In epithelioid sarcoma, especially in proximaltype epithelioid sarcoma, increased cell disadhesion
and motility by dysadherin plays an important role
to acquire aggressive biological behavior. However,
in malignant rhabdoid tumor, cell growth cycle that
is characterized and regulated by hSNF5/INI1 gene
abnormalities seems to be critical to lethal biological
behavior rather than dysadherin.
Acknowledgements
This work was supported in part by Grants-in-Aid
for Scientific Research from the Japan Society for
the Promotion of Science (15590304, 17659103),
Tokyo, Japan. We are grateful to Miss Naomi Tateishi,
Miss Yoko Nozuka and Mr Naoto Koyama for
their excellent technical assistance. We thank Miss
Katherine Miller (Royal English Language Centre,
Fukuoka, Japan) for revising the English used in this
article.
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